A new sub-family of H-2 class I genes has been identified in the genome of the C57BL/1O mouse. The family consists of at least two genes which have not been previously identified. One of these, Mb1, has now been extensively characterized. Using a series of recombinant strains of mice, and taking advantage of restriction enzyme polymorphisms, it has been possible to map the Mb1 gene to the right of the Qa locus. DNA sequence analysis of Mb1 demonstrates that the exon organization of this gene resembles that of other class I genes. Furthermore, it is capable of encoding a transmembrane protein with a structure similar to other class I molecules. However, the level of DNA sequence homology of Mb1 to other H-2 genes is no greater than to either human, pig, or rabbit. Furthermore, the over-all organization of Mb1 is more similar to man and pig than to mouse. Whereas all previously reported H-2 class I genes have third introns of 1.2-2 kb, Mb1 has an intron of 600 bp, similar to those of human and porcine class I genes. Taken together, these data suggest that Mb1 may represent a direct descendant of a primordial class I gene, which antedates speciation. In support of this conclusion is the observation that a variety of wild mice, representing millions of years of evolutionary divergence, contain Mb1 in their genomes. Although other members of the Mb1 family appear to be expressed, no direct evidence for Mb1 expression has been obtained, despite the fact that it is a structurally competent gene.